J. Exp. Biol. (1965), •>. 463-474
With 7 text-figures
Printed m Great Britain
463
THE ELECTRICAL POTENTIAL DIFFERENCE ACROSS THE
EPITHELIUM OF ISOLATED GILLS OF THE CRAYFISH
AUSTROPOTAMOBIUS PALLIPES (LEREBOULLET)
BY P. C. CROGHAN,* R. A. CURRAf AND A. P. M. LOCKWOODJ
Department of Biophysics, University of Edinburgh
(Received 7 October 1964)
INTRODUCTION
There has been a considerable amount of work on the electrical potential differences
and active transport processes of ions across various vertebrate epithelia (Ussing,
i960). Little work of this type has been attempted on invertebrate material. As the
crustacean gill epithelium has only a single layer of cells separating the body fluid and
the external medium, it should be particularly suitable for such studies. Crustacea
living in hypo-osmotic media take up NaCl from the medium and it has been assumed
that this uptake is across the gill epithelium. In the case of Eriocheir it has been shown
that isolated gills can take up NaCl rapidly from a dilute NaCl solution (Koch, 1954).
There have been measurements of the electrical potential difference between the
haemolymph and dilute outer medium in whole Austropotamobius. The values range
from —4-1 to — 6-6 mV. in tap water (Bryan, i960) and — 5 to — 47 mV. in a medium
of 0-3 mM/1. KC1 (Shaw, i960). The sign of the potential is that of the haemolymph
with respect to the medium. These results indicate that both chloride and sodium
must be actively transported into the haemolymph against an electrochemical potential
difference. In this paper measurements of the electrical potential difference across
the epithelium of isolated perfused gills of Austropotamobius pallipes are reported.
THEORETICAL CONSIDERATIONS
The total electrical potential difference across the epithelium between the outer
medium and the inner medium (body fluid) measured using concentrated KC1
solution bridges will be termed epithelial potential (EE). Using the sign convention
adopted here it defines the electrical potential of the body fluid with respect to the
outer medium. The epithelial potential is the sum of the membrane potentials of the
outer and inner epithelial cell membranes.
If an ion j is in passive equilibrium between two phases (e.g. body fluid and outer
medium), the electrical potential difference between the two phases (Ej) is given by
the Nernst equation
Present address:
• School of Biological Sciences, University of East Anglia.
t Instiruto Oceanografico, Universidad de Oriente, Venezuela.
X Department of Oceanography, University of Southampton.
464
P. C CROGHAN, R. A. CURRA AND A. P. M. LOCKWOOD
where C^ and Cf are the concentrations (strictly activities) of ion j in the two phases.
The extent to which the calculated Nernst potential for ion / differs from the actual
epithelial potential indicates the extent to which ionj is out of equilibrium. A fuller
discussion of this is given by Dainty (1962).
An actual membrane potential is usually regarded as a diffusion potential and can
be related to ionic concentration differences and selective permeability properties of
the membrane. A fuller discussion of this is also given by Dainty (1962). Information
concerning these permeability properties can be obtained if the change of membrane
potential following a change to a new medium on one side of the membrane is determined. One approach that has been used is to apply the Goldman constant field
equation, but this equation cannot be applied in this study of this gill epithelium where
there are two cell membranes in series as there is no information concerning the
ionic concentrations in the epithelial cell fluid. A different approach is to consider
the transport numbers of the ions in the cell membranes. The approach given below
is an extension of that given by Hodgkin and Horowicz (1959). An expression for
the membrane potential for an epithelium can be written
EE = YiT°jE° + ?iTIiEl
y-i
(2)
y-i
where T] is the transport number of ionj in the outer (O) and inner (I) cell membrane
and Ef the Nernst potential of the ion across the outer (O) and inner (7) cell membrane.
If the concentration of a single permeable (electrogenic) ion i is varied in the outer
medium and the 'instantaneous' potential change is determined (i.e. before changes
in the ionic concentrations in the cell fluid have occurred), then
dE
B _ Tg , Y v
If the transport numbers are constants or if all the ions to which the membrane is
significantly permeable are in equilibrium across the cell membrane (E$ — E^), then
the equation simplifies to
Further if finite changes are considered
Strictly this integration is only approximate as the transport number of an ion in a
membrane will be a function of the concentration of the ion in the membrane. Also
the ions are unlikely to be in equilibrium across the outer cell membrane of an
epithelium across which ions are being transported. In view of this, the transport
numbers defined by equation (5) should really be termed apparent transport numbers.
They should give valuable information concerning the relative ease with which an ion
can cross passively the outer cell membrane.
The permeability coefficient and transport number of an ion in a membrane are
of course related. The relationship is considered by Hodgkin & Horowicz (1959).
Gills of the crayfish
465
MATERIAL AND METHODS
Specimens of the British crayfish Austropotamobius palHpes were kept in a medium
of crayfish Ringer solution diluted to o-oi Ringer and to which was added a little
solid CaCO3. The animals were kept in this medium in large aerated tanks at 9-10° C.
and survived excellently. Intermoult animals only were used. In fact the relatively
low temperature virtually suppressed moulting. Only the podobranch gills from the
2nd maxilliped to 4th pereiopod inclusive were used in these experiments. Whole gills
were cut off between the basal plate and the insertion on the appendage and placed
in crayfish Ringer solution until required.
Experimental
solution out
Experimental
solution in
Multiway tap
Fig. 1. Diagram of apparatus for determining the epithelial potential of a perfused gill in
various external media.
In the experiments reported here the gill was continuously perfused with Ringer
solution (van Harreveld, 1936) containing 1 mg. Evans Blue/100 ml. The apparatus
is represented diagrammatically in Fig. 1. The Ringer solution was contained in an
all-glass syringe driven by a Palmer slow injection apparatus. The cannulae were
made by drawing out polythene cannula tubing. The afferent cannula with a tip
diameter of ca. 0-2 mm. was inserted into the afferent vessel and the efferent cannula
with a tip diameter of ca. 0-5 mm. was inserted into the large efferent vessel. The
cannulae were held in place by a fine nylon ligature just below the basal plate. The
gill was perfused at ca. o-4/il./sec. The dye was added to check that the gill filaments
were properly perfused.
The medium was held in a small funnel {ca. 5-5 ml. capacity) connected by a multiway tap to 5 1. polythene reservoirs containing aerated solutions. The gill was lowered
into the medium until the filaments were immersed. During the experiments the
466
P. C. CROGHAN, R. A. CURRA AND A. P. M. LOCKWOOD
medium was flowing continuously at ca. 1 ml./sec. The multiway tap enabled the
solution to be changed rapidly.
The electrodes were standard Pye Hg-calomel-sat. KC1 electrodes (type 11161).
.
£-60
ml
J,
the Ual
c
a -20
w
-
0
2M
3M
1P
2P
Podobranch insertion
3P
4P
Fig. 2. Epithelial potential (mean ± s.D. ± s.E.) compared with position of gill in gill series.
Outer medium o - oi Ringer solution. Sign of potential is that of body fluid with respect
to medium. Podobranch insertion: 2 M, 3 M , maxillipeds; 1P (chela), 2 P, 3 P, 4 P, pereiopods.
-100 r
-80
-60
•a
•a
§
8.
•a
-40
-20
0
10
I
1
0-1
0-01
External Ringer concentration
0-001
Fig. 3. Epithelial potential (mean ±s.D. ± s.E.) in various external concentrations of Ringer
solution. Sign of potential is that of body fluid with respect to medium.
Gills of the crayfish
467
One electrode was inserted in the afferent line. The reference electrode was attached
by a rubber collar to a 2 mm. diameter glass tube drawn out to a fine rounded off tip
containing sat. KC1 agar, and was placed in the medium. When not in use the electrode
tips were kept immersed in sat. KC1 solution. The electrodes were connected to the
input of a Pye Master pH meter-millivoltmeter and the output of this was connected
via a 100 Q. potentiometer to a Kipp BDi Micrograph pen recorder. Both sides of
the input were isolated from earth. After each experiment the gill was removed and
the zero was taken by immersing the afferent cannula and reference electrode in
Ringer solution. With some reference electrode tips there was instability and shift of
potential when the flow was turned on. This was particularly noticeable with the
most dilute external solutions. Care was taken to select an electrode tip in which
this effect was minimal. The experiments were carried out at room temperature
{ca. 17° C ) .
Table 1. Composition of some experimental solutions
Ionic concentration (mM./l.)
Solution
Ringer (R)
A (001 R)
B
Na
207
2-07
10-4
C
207
D
2-07
2-07
2-17
E
F
K
Cl
5'4
0054
0054
0-27
0054
0054
0054
243
243
2-43
2-43
12-2
2-43
2-43
Ca
13-5
OT35
OI35
0-135
O-I35
1-35
0-135
HCO,
2-4
0-034
0-024
0024
0024
0024
0-12
Mg
BS
Ch
a-6
0-026
0-026
0-026
0-026
0-026
0-026
—
—
8-3
0-216
—
—
—
—
9-7:
—
—
—
1-22
BS is benzene-sulphonate and Ch is choline.
RESULTS
In all the experiments the electrical potential was measured with o-oi Ringer, the
medium in which the animals had previously been kept, flowing past the gill. The
results are summarized in Fig. 2. The mean epithelial potential and standard deviation
for all the gills in this medium is —60+12 mV. (40 gills from 13 animals).
In one group of experiments (13 gills from 4 animals) the solution flowing past the
gill was changed as follows: Ringer ( R ) - O - I R - O - O I R - O - O O I R - O - O I R - O - I R - R . The
potential was taken when it had reached a steady value ( < 30 sec.) and the mean
value was taken when there were two measurements. The results are summarized in
Fig. 3In the other group of experiments (27 gills from 9 animals) the concentration of
individual physiologically important ions were varied in turn using benzene-sulphonate
or choline as assumed non-permeating and therefore non-electrogenic counter ions.
The solutions used are given in Table 1. The concentrations of univalent ions were
varied by x 5 and that of calcium by x i o . The potential was recorded with solution A
(o-oi R) flowing until a steady value was obtained, and then the solution was changed
to one of the experimental solutions. The solutions were changed as follows:
A-B-A-C-A-D-A-E-A-F-A.
Some examples of records are given in Fig. 4.
468
P. C. CROGHAN, R. A. CURRA AND A. P. M. LOCKWOOD
The 'instantaneous' change of potential was taken as the change in 10 sec. after
the recorder pen had begun to move. The results expressed as apparent transport
numbers are summarized in Figs. 5, 6 and 7.
-100
-60
a
-20
-100
-80
8.
-40
-20
8
7
6
5
4
3
2
1
0
Minutes
Fig. 4. Examples of recordings of epithelial potentials. Letters refer to the solutions flowing
(see Table 1). Upper record is a 3rd maxilliped podobranch and lower record is a and
pereiopod podobranch.
DISCUSSION
It is considered that the main advantage of using a preparation perfused with
Ringer solution is that the ionic concentrations are known precisely and experimental
variations due to individual variation in haemolymph composition are avoided. As
the Ringer solution is very similar to the haemolymph composition (Lockwood, 1961)
the conclusions drawn from these experiments are considered relevant for the normal
gill.
The results summarized in Fig. 2 show that in all cases the body fluid is considerably negative with respect to the medium. The potential differences are considerably
larger than those of Bryan (i960) for whole animals, suggesting possibly that short
circuiting had occurred in his experiments. There appears to be no significant dif-
Gills of the crayfish
469
ference between the mean values of the epithelial potential of gills from different
positions in the gill series. Although significant differences were frequently found
between individual gills taken from a single animal, it should be remembered that in
an intact animal the potential difference across all the gills will tend to be clamped at
a similar value by the presence of common conducting media on each side of the
epithelium.
07
r
ft*
Na
' .-V:
0
07
K
•
••••••• «
•
0
07 i Ca
•
• ••
c
e
|o
S 0-7
Cl
§
a
0
07
HCOj
• *
•
••• •
-100
Epithelial potential (mV.)
Fig. 5. Apparent transport numbers of ions in relation to the epithelial potential immediately
preceding the experimental determination.
The effect of the gill epithelium on the potential differences between Ringer solution
and o-oi R is even clearer when it is realized that the diffusion potential between
these two solutions without any membrane is about + 26 mV.
It is simple to compare the Nernst potentials of the individual ions with the epithelial
potential of Austropotamobius gills as the concentration of each ion differs by x 100
as between the two solutions. The results are:
Mean epithelial
potential (mV)
EE
-60
Nernst potential (mV)
-n6
-116
-58
-58
+116
+116
Exp. BloL 42, 3
470
P- C. CROGHAN, R. A. CURRA AND A. P. M. LOCKWOOD
o-7
r
Na
c
a 07
r
HCO,
I
0-5
0
Apparent transport number of chloride
0-5
Fig. 6. Relation between apparent transport numbers of the other ions and the apparent
transport number of chloride.
0-6
o
a
\
i
aa.
<
i
J_
2M
3M
1P
2P
Podobranch insertion
3P
_J
4P
FIR. 7. Relation between the apparent transport numbers (mean ± S.E.) of sodium and chloride
and position of gill in gill series. Sodium, closed circles; chloride open circles. Podobranch
insertion: i M , 3M, maxillipeda; i P , aP, 3P, 4P, pereiopods.
Gills of the crayfish
471
Clearly, the electrochemical potential of chloride in the body fluid is very much
greater than that in the external medium. The electrochemical potentials of sodium
and potassium are also considerably above those in the medium. All these ions must
therefore be actively transported from the medium into the body fluid across the gill
epithelium. The divalent ions appear to be more or less in electrochemical equilibrium
and thus there is no evidence to imply active transport. No conclusion will be drawn
in the case of bicarbonate as there is no information concerning the bicarbonate
concentration in the haemolymph which may be considerably different from that in
the Ringer solution.
A similar approach to that described above has been applied by House (1963) in
considering active transport across the gill epithelium of the brackish-water teleost
Blenmus pholis. But there are several papers purporting to decide the extent to which
the ionic concentrations in the body fluid differ from equilibrium with the medium
in which no attempt was made to measure the epithelial potential (Robertson, 1957,
i960; Webb, 1940). Conclusions arrived at in this way are fallacious.
The steady values of potential difference summarized in Fig. 3 show that the potential varies considerably in different external concentrations of Ringer solution. The
potential difference was approximately zero with Ringer solution on both sides of
the epithelium. This would be expected if the epithelium were a single membrane,
and perhaps it suggests that under these conditions the composition of the cell fluid
is similar to that of the Ringer solution. In more dilute external solutions the epithelial
potential appears to rise to a maximum in o-oi R. Although this maximum is not
statistically significant when the mean data are considered, a maximum was in fact
shown by a significant number of the individual gills (8 out of 13 gills) from which
the data in Fig. 3 were derived. There was apparently no correlation between the type
of response and the position of the gill in the gill series. This suggests that in external
media more concentrated than o-oi R, the epithelium (considered as a single membrane)
is behaving as a selectively cation-permeable membrane, and that in media more
dilute than about o-oi R some change occurs in the permeability properties. The
epithelial potential is maximal in the medium to which the animal had previously
been adapted, and at this point the potential is relatively insensitive to changes in
external concentration. The significance of this is uncertain.
The situation in the crayfish gill described above contrasts sharply with that found
in amphibian skin. In frog skin the inside solution is normally positive with respect
to the external solution (Steinbach, 1933; Ussing, i960). Considered as a single
membrane frog skin appears to behave as a selectively chloride-permeable membrane.
Normally in frog skin the uptake of chloride appears to be a purely passive movement
down an electrochemical potential gradient. However, in certain cases, particularly
when the external solution is 3 mM./l. KC1 solution, there is also evidence of an
active uptake of chloride across anuran skin (Jorgensen, Levi & Zerahn, 1954).
Another case where there is an active transport of chloride across an external epithelium is that of the gill epithelium of the brackish-water teleost Blenmus pholis
(House, 1963).
The results of the experiments in which the outer concentration of an (assumed)
single permeable ion was changed are difficult to interpret. Due to the complex
filamentous and lamellar structure of the gills it is probably impossible to obtain a
30-2
472
P. C. CROGHAN, R. A. CURRA AND A. P. M. LOCKWOOD
very rapid change of the medium at the outer surface of the epithelial cells even with
the fast flow rates used. Furthermore, as the epithelial cells are thin, varying from
ca. i/i up to as much as 30/i around nucleus (paraffin sections after fixation in 1 %
OsO4 in Ringer solution), rapid changes in the cell fluid composition could occur
The 'instantaneous' potential change was in fact taken as the change in 10 sec. after
the pen had begun to move. In a number of cases the potential had reached, or nearly
reached, a steady value in this time (an exception to this being solution £(Ca) where
the potential usually changed more gradually). Thus even if the steady potential
values in the various media had been used in place of the 10 sec. values the appearance
of Fig. 5 would be little different.
In fact no clear picture emerges from Fig. 5. There is no correlation between the
membrane potential with o-oi R outside and the transport number of any ion in the
outer cell membrane. This is rather surprising. It must be remembered that our
epithelial potential is the sum of the outer and inner cell membrane potentials, and
the results may suggest that the outer cell membrane potential has a much lower
value than the inner cell membrane potential, which would then act as the major
determinant of the overall epithelial potential and effectively randomize the relation
plotted in Fig. 5. Another point to be considered in the interpretation of Fig. 5 is
that the gill filaments are divided into two populations with different histochemical
properties (Curra & Croghan, in preparation) and the electrical measurements were
made on these two populations. A point worth making is that the lower potentials
are not associated with damaged leaking gills as the transport numbers would be much
lower if short-circuiting had occurred.
The transport numbers indicate that the gills are mainly cation-permeable (especially
to Na), which is in keeping with the results obtained with various dilutions of Ringer
solution outside the gills. It should be stressed that these transport numbers apply
to an outer medium of o-oi R. As a transport number is a function of the concentration
of the ion in the membrane, the values must be considered in relation to the absolute
values of the concentration of the ion on either side of the membrane and may change
considerably in different external media.
If we consider the data of Fig. 5 in terms of individual gills correlations do become
apparent. Fig. 6 indicates a marked inverse correlation particularly of the transport
numbers of sodium and calcium with the chloride transport number. An inverse
correlation would be expected as it is a property of transport numbers that
N
2 TJ = 1. The mean value and standard deviation of the sum of the transport
y-i
numbers of the ions considered in Fig. 6 is 0-97 ± 0-2. There appears to be a continuous gradation of gill properties from a mainly cation-permeable towards a more
chloride-permeable gill type. Also Fig. 7 indicates that the transport numbers of the
two major ions, sodium and chloride, are correlated with the position of the gill in the
gill series. The anterior gills contrast sharply with the posterior gills. No regular correlation was found with calcium. The significance of the difference of properties between
the anterior and posterior podobranchs is uncertain. It can be mentioned, however,
that Koch, Evans & Schicks (1954) found that the three posterior gills of Eriocheir
appear to take up sodium more rapidly than the anterior gills.
These results may be compared with those found in other material. Hodgkin &
Gills of the crayfish
473
Horowicz (1959) were able to determine the transport numbers of potassium and
chloride in the cell membrane of single muscle fibres. However, this study was
simplified because these ions are in electrochemical equilibrium (at least approximately)
in muscle cells (JEK = E^) and these are the only ions to which the membrane is
appreciably permeable. In these circumstances equation (4) holds exactly. Further,
because of the simple geometry of their preparation, Hodgkin & Horowicz (1959,
i960) were able to get a virtually instantaneous change to a new medium. In experiments where the internal composition could vary there were drifts of membrane
potential over long periods. Results of this precision, however, can scarcely be
expected in such a complex organ as the crayfish gill. Experiments involving changes
in the composition of the bathing solutions have also been carried out on frog skin
(Koefoed-Johnsen & Ussing, 1958). Here, however, only the steady values of the
potentials after equilibration in various media are given and the possible effects of
variations of the concentration in the cell fluids are ignored, in spite of the fact that
variations of the volume of the epithelial cells were observed in some cases. Furthermore these experiments were made under conditions where chloride conductance had
been artificially reduced. Nevertheless, their conclusions concerning the relative
permeability of the outer and inner membranes to sodium and potassium have been
confirmed by MacRobbie & Ussing (1961) using more critical methods.
It is tempting to try to construct a model for the epithelial cell of the gills of Austropotamobius similar to that developed in the above-mentioned papers for the epithelial
cell of frog skin, and in particular to decide in which membranes the various pumps
are situated. Such an attempt is really premature. But in general it can be considered
logical that a specific ion pump will only be found in a membrane relatively impermeable to that ion. Thus considering sodium and chloride, the two major ions in the
medium (o-oi R), the transport number of chloride in the outer cell membrane is in
the majority of gills (particularly the posterior gills) significantly less than the transport
number of sodium (Figs. 6, 7). Thus tentatively the chloride pump could be placed
in the outer cell membrane and, by analogy with frog skin, the sodium pump in the
inner cell membrane.
However, these and related problems need to be investigated more critically using
other techniques. Valuable information could be obtained by studying the effects of
changes of the internal medium, which is technically possible with the perfused gill
preparation. Even more valuable would be to obtain information on the chemical
composition of the epithelial cell fluid and to split the overall epithelial potential
into separate outer and inner membrane potentials by introducing a micro-electrode
tip into the epithelial cell.
SUMMARY
1. A technique is described for recording the electrical potential differences across
the epithelium (epithelial potential) of isolated podobranch gills of Austropotamobius
pallipes continuously perfused with Ringer solution in various external media.
2. In a medium of o-oi Ringer, in which the animals had previously been kept,
the mean epithelial potential ± standard deviation was — 60 ± 12 mV. (Sign defines
potential of body fluid with respect to external medium.) Chloride, sodium and
potassium must be actively transported into the body fluid against an electrochemical
gradient. Calcium and magnesium ions appear to be approximately in equilibrium.
474
P- C- CROGHAN, R. A. CURRA AND A. P. M . LOCKWOOD
3. The steady-state membrane potentials were recorded in various external concentrations of Ringer solution. The potential is about zero with Ringer solution outside
and rises to a maximum with 001 Ringer outside.
4. Changes of the electrical potential were recorded when the concentration of a
single electrogenic ion was changed in the external medium (o-oi Ringer), and were
used to define an apparent transport number of the ion in the outer cell membrane.
5. There was no correlation between the transport numbers and the epithelial
potential.
6. There was a continuous gradation of gill types from a predominantly cationpermeable type towards a more chloride permeable type. There is a correlation between
the type of gill and the position in the gill series.
7. The properties of the epithelial cells of Austropotamobius gill are significantly
different from those of the epithelial cells of frog skin. It is suggested that in Austropotamobius a chloride pump is situated in the outer cell membrane.
We wish to thank Prof. J. Dainty for many helpful discussions. We also wish to
thank the Medical Research Council for a grant to one of us (P. C. C.) for equipment.
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Addendum. A recent paper by Biehwski, J. (1964), 'Chloride transport and water intake into
isolated gills of crayfish' (Comp. Biochem. Physiol. 13, 423), has demonstrated directly a net
transport of chloride from dilute outer medium into the haemolymph in isolated crayfish gills.